Cholesterol-Modified Black Phosphorus Nanospheres for the First NIR

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Biological and Medical Applications of Materials and Interfaces

Cholesterol-Modified Black Phosphorus Nanospheres for the First NIR-II Fluorescence Bioimaging Yifan Xu, Feng Ren, Hanghang Liu, Hao Zhang, Yaobao Han, Zheng Liu, Wenliang Wang, Qiao Sun, Chongjun Zhao, and Zhen Li ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b05825 • Publication Date (Web): 23 May 2019 Downloaded from http://pubs.acs.org on May 25, 2019

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ACS Applied Materials & Interfaces

Cholesterol-Modified Black Phosphorus Nanospheres for the First NIR-II Fluorescence Bioimaging Yifan Xu†, ‡, Feng Ren‡, Hanghang Liu‡, Hao Zhang‡, Yaobao Han‡, Zheng Liu‡, Wenliang Wang‡, Qiao Sun‡, Chongjun Zhao†, *, Zhen Li‡, * Key Laboratory for Ultrafine Materials of Ministry of Education, Shanghai Key Laboratory of Advanced Polymeric Materials, School of Material Science and Engineering, East China University of Science and Technology, Shanghai, 200237, China. *E-mail: [email protected] †

‡Center

for Molecular Imaging and Nuclear Medicine, State Key Laboratory of Radiation Medicine and Protection, School for Radiological and Interdisciplinary Sciences (RAD-X), Soochow University, Collaborative Innovation Center of Radiation Medicine of Jiangsu Higher Education Institutions Suzhou 215123, China *E-mail: [email protected]

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ABSTRACT: Black phosphorus (BP) nanostructures with unique layer-dependent properties have been extensively applied in the fields of electronic devices, energy conversion and storage, and nanomedicine. As a narrow bandgap semiconductor, they are expected to show the strong second near-infrared (NIR-II) fluorescence. However, there is no report on the NIR-II fluorescence of free-standing BP nanostructures, which have great potential in the NIR-II fluorescence bioimaging due to their excellent biocompatibility and biodegradability. Here, for the first time, we report that the BP nanoparticles modified with cholesterol exhibit strong NIR-II fluorescence, and can be encapsulated with PEGylated lipid to form BP@lipid-PEG nanospheres for in vitro and in vivo NIR-II imaging. The resultant BP@lipid-PEG nanospheres exhibit broad emissions from 900 to 1650 nm under excitation by an 808 nm laser, and have 8% quantum yield of that of standard dye IR26. We also show that NIR-II fluorescence image acquired with emission beyond 1400 nm has the sharpest contrast and can be used to in situ measure the diameter of blood vessels. In addition to NIR-II fluorescence imaging, we also show the potential of BP@lipid-PEG nanospheres in photoacoustic imaging. Both the long-wavelength NIR-II fluorescence imaging and PA imaging reveal that the as-fabricated BP@lipid-PEG nanospheres can be gradually metabolized by liver in 48 h, thus making them promising for bioapplications. KEYWORDS: black phosphorus, cholesterol-modification, nanospheres, NIR-II fluorescence, bioimaging

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1. INTRODUCTION Nanoscale black phosphorus (BP) as an emerging two-dimensional (2D) semiconductor with tunable band gap and high carrier mobility has attracted considerable interests from the fields of transistor, photocatalysis, photodetector, energy storage and conversion.1-5 Naturally rapid degradation of BP nanostructures in ambient environment deteriorates their performance, however, which has been improved by covalent and coordinative modification, and physical encapsulation.6-9 Recently, two-dimension BP nanostructures have been exploited for bio-applications due to their intrinsically high extinction coefficient, excellent photothermal conversion efficiency, and large surface-area-to-volume ratio.8 For example, BP nanostructures were used for photoacoustic (PA) imaging and photothermal therapy of tumor under irradiation of near-infrared (NIR) light.7-11 Compared with traditional ultrasonic imaging, PA imaging can provide more sensitive, higher contrast and spatial resolution tomographic images of tissue, and can monitor bio-distribution and pharmacokinetics of nanodrugs to determine the optimal therapy time.12-19 BP nanostructures were also encapsulated into drug delivery systems to control the release of drugs in tumor through their photothermal effect.20-23 They were further decorated with functional moieties such as catalysts and photosensitizers to show excellent photodynamic therapy of tumor under

the

irradiation

of

NIR

light.24-29

Synergistic

photothermal/photodynamic/chemical/gene/immuno therapy has been performed with BP nanostructures.30-33 Recently they were incorporated into scaffolds and employed for bone regeneration by boosting osteogenic differentiation of human stem cells.34, 35 3 / 29



The above examples demonstrate the great potential of BP nanostructures in biomedical field. As a narrow bandgap semiconductor, BP nanostructures are expected to show strong fluorescence in the second near-infrared (NIR-II) window, which has been observed in few-layer BP with a horizontal size ranging from 130 nm to 50 μm on SiO2/Si substrate.36-40 However, there is no report on the NIR-II fluorescence of freestanding BP nanostructures and their application for in vivo NIR-II imaging. Compared with visible and NIR fluorescence imaging, the NIR-II fluorescence imaging affords higher spatial resolution, deeper penetration, lower optical absorption and scattering from biological tissues, and lower autofluorescence.41-43 The long-wavelength NIR-II imaging can improve the diagnosis of tumor for better treatment due to the deep penetration and sharp contrast.44 Here, for the first time, we show the NIR-II fluorescence of free-standing BP nanospheres and their application for in vivo NIR-II imaging. The BP nanospheres consist of tens of cholesterol-modified BP nanoparticles with an average size of 20 nm, and can strongly emit NIR-II fluorescence from 900 to 1650 nm under excitation by an 808 nm laser. By setting different wavelength optical filters, we successfully used them to measure the diameter of a tissue-covered capillary with high accuracy through the NIR-II fluorescence imaging, and used to in situ measure blood vessels in legs of mice clearly with a high signal-to-noise ratio. The in vivo NIR-II fluorescence imaging was carried out to investigate the accumulation and metabolism of BP nanospheres in liver by real-time monitoring the NIR-II fluorescence signal, and the result is matched well with that of PA imaging. Our work demonstrates the great potential of BP nanoparticles 4 / 29


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serving as a new biocompatible and biodegradable imaging probe for NIR-II fluorescence imaging with high sensitivity and deep penetration.

2. EXPERIMENTAL PROCEDURES 2.1 Materials Red phosphorus (RP) was purchased from Aladdin Reagents. Cholesterol (Chol) and L-a-Lecithin were purchased from Aladdin Reagents and Solarbio Science & Technology

Co.,

Ltd.,

and

DSPE-mPEG2K

(1,2-distearoyl-sn-glycero-3-

phpsphoethanolamine-N-[methoxy (polyethylene glycol)-2000]) was obtained from Nippon Fine Chemical Co., Ltd., Tetrahydrofuran (THF) was purchased from Yonghua Chemical Technology (Jiangsu) Co., Ltd. 2.2 Synthesis of BP-Chol nanoparticles The BP-Chol nanoparticles were prepared from RP powder and cholesterol by a solventless high energy mechanical milling (HEMM) approach.8 Briefly, RP powder was milled by stainless steel balls in a hardened steel vial with a capacity of 100 mL for 96 h. Then cholesterol was added to modify nanoparticles, and the reaction mixture was milled for 8 h. Finally, the product was dispersed in THF, and then the supernatant containing BP nanoparticles was collected by centrifugation and stored at -20 °C for further use. 2.3 Synthesis of BP@lipid-PEG nanospheres The BP@lipid-PEG nanospheres were prepared as follows. The THF solution of BPChol nanoparticles, DSPE-mPEG2K and L-a-Lecithin were placed in a fume cupboard to remove THF, and then mixed them with Milli-Q water under vigorous stirring. The 5 / 29



mixture solution was centrifuged at 3000 rpm to remove precipitates, the supernatant containing BP@lipid-PEG nanospheres was collected for further use. 2.4 Characterization BP powder and BP-Chol nanoparticles were analyzed by a Shimadzu XRD-6000 Xray diffractometer (XRD) using Cu-Kα1 (0.15406 nm) radiation to determine their crystal structures. A FEI Tecnai G20 transmission electron microscope (TEM) working at 200 kV was used to characterize the size and morphology of BP nanoparticles and nanospheres. The UV–vis–NIR absorbance spectra of BP@lipid-PEG nanospheres were recorded on a PerkinElmer Lambda 750 UV–vis–NIR spectrophotometer. Fluorescence spectra of BP@lipid-PEG nanospheres were conducted on a FLS980 spectrometer (Edinburgh Instruments, UK). A Veeco atomic force microscope (AFM) was utilized to measure the size and thickness of BP-Chol nanoparticles. Hydrodynamic size of BP@lipid-PEG nanospheres was measured with a Malvern Zetasizer Nano ZS90 at 25 °C equipped with a solid state He–Ne laser (λ = 633 nm). 2.5 Photothermal performance of BP@lipid-PEG nanospheres Different concentrations of aqueous solutions of BP@lipid-PEG nanospheres were irradiated under an 808 nm laser with a power density of 1.0 W cm-2 for 10 min. The temperatures of BP@lipid-PEG solutions were recorded with an infrared (IR) thermal camera. Their photostability was tested by 5 cycles of irradiating and cooling processes. 2.6 In vitro and in vivo photoluminescence imaging in the NIR-II window A NIR-II Imaging System (Serious II 900 – 1700) manufactured by Suzhou NIROptics Co., Ltd. (China) was used to record the in vitro and in vivo NIR-II fluorescence 6 / 29


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images. For in vitro imaging, an aqueous solution of BP nanospheres (400 µg mL-1) was loaded into a glass capillary (diameter = 0.41 mm) and covered with different thickness of chicken slices (1 mm, 2 mm, and 3 mm). The images of capillary were captured by selecting different wavelength filters under excitation by an 808 nm laser. The in vivo images were collected after BP nanospheres aqueous solution (150 μL, 1.3 mg mL-1) was intravenously injected into normal nude mice anesthetized with 1.5% isoflurane. The power density of the 808 nm laser was 0.25 W cm-2. 2.7 Photoacoustic (PA) imaging A real-time multispectral photoacoustic tomographic imaging system (MSOT, inSight/inVision 256, iThera Medical GMbH) was employed to do in vitro and in vivo PA imaging. For in vitro PA imaging, different concentrations of aqueous solutions of BP@lipid-PEG nanospheres (e.g., 0, 15.6, 31.3, 62.5, 125, 250 and 500 μg mL-1) were excited with 680 nm laser to acquire PA images. For in vivo PA imaging, a normal nude mouse anesthetized with 1.5% isoflurane beforehand was intravenously injected with BP@lipid-PEG nanospheres aqueous solution (150 μL, 1.3 mg mL-1), PA images were acquired before and post injection of 10 min, 20 min, 30 min, 1 h, 2 h, 4 h, 8 h, 24 h and 48 h. After the end of PA imaging, the mouse was sacrificed to collect its heart, liver, spleen, lung, kidney, intestines and stomach for observing with the NIR-II Imaging System (808 nm laser, 1250 nm optical filter).

3. RESULSTS AND DISCUSSIONS 3.1 Preparation and characterization of BP@lipid-PEG nanospheres 7 / 29



The BP@lipid-PEG nanospheres were prepared by slow evaporation of THF solution of BP-Chol nanoparticles, DSPE-mPEG2K and L-a-Lecithin, and then dissolved in Milli-Q water. BP-Chol nanoparticles were slowly assembled into spheres and encapsulated by DSPE-mPEG2K and L-a-Lecithin during the solvent evaporation (Fig. 1a). TEM and AFM were employed to characterize the morphology and size of BPChol nanoparticles (Fig. 1b, c). The average horizontal size of BP-Chol nanoparticles was around 20 nm. The height of BP-Chol nanoparticles was 4.6 ±1.9 nm. Assuming the thickness of a single layer of BP is 0.63 nm, we could estimate that each BP-Chol nanoparticle was composed by 4-10 layers of BP.45 The crystal structures of the as-prepared BP and BP-Chol nanoparticles were characterized by XRD. As shown in Fig. 1d, all the diffraction peaks are consistent with those of standard orthorhombic black phosphorus (JCPDS No. 76-1957), and their position and strength do not show obvious difference before and after modification. The result demonstrates the negligible influence of cholesterol modification on crystal structure and size of BP nanoparticles. The Fourier transform infrared (FTIR) spectra in Fig. S1a clearly show the characteristic C-H vibration at ~2900 cm-1 and ~1400 cm1

in the BP-Chol sample, which are similar to those of pure cholesterol. The broad

absorption bands at ~996 cm-1, ~1131 cm-1 and ~1670 cm-1 can be ascribed to the P-O stretching and P=O stretching modes. These results verify that BP nanoparticles were successfully modified by cholesterol. As displayed in Fig. 1e-f and Fig. S2, BP@lipid-PEG nanospheres have an average size of 97.7 ± 27.8 nm. The hydrodynamic sizes of BP-Chol nanoparticles and 8 / 29


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BP@lipid-PEG nanospheres were measured to be 42.1 nm and 145.9 nm (Fig. S1b), respectively. Furthermore, the high-resolution TEM (HRTEM) was used to characterize the crystal structure of nanospheres, the observed interplanar distance 0.262 nm is well consistent with that of (040) plane of black phosphorous (Fig. 1e, inset). 3.2 NIR-II fluorescence and photothermal properties of BP@lipid-PEG nanospheres Fluorescence images of BP@lipid-PEG nanospheres (1.3 mg mL-1) were captured with the NIR II Imaging System under 808 nm laser irradiation (Fig. 2a). Without NIR irradiation, BP@lipid-PEG nanospheres aqueous solution was brown. Under the NIR irradiation, the nanosphere solution exhibited strong photoluminescence, even it was filtered by different longpass wavelength of filters such as 810 nm, 1000 nm, 1250 nm, and 1400 nm. This result indicates a broad emission of BP@lipid-PEG nanospheres in the NIR-II region. The NIR-II fluorescence of different concentrations of BP@lipid-PEG nanosphere solutions was investigated. As displayed in Fig. 2b, broad emissions were observed in all spectra of BP@lipid-PEG nanosphere solutions. Two strong peaks at ~1076 and ~1288 nm in the NIR-IIa region and one weak peak at ~1596 nm in the NIR-IIb region can be attributed to different thicknesses of BP-Chol nanoparticles (Fig. 1c).46 The fluorescence intensity of BP@lipid-PEG nanosphere solutions increased gradually with the increase of their concentration. In addition, their intensities at 1070 nm were linearly increased with concentration increasing from 35.0 to 132.1 µg mL-1. Further increasing concentration led to the deviation of linearship due to the partial quenching of 9 / 29



fluorescence at high concentration (Fig. 2c). UV-vis-NIR spectra of BP@lipid-PEG solutions with different concentrations were also measured and shown in Fig. S3a. An abroad absorption in the NIR window was observed and their absorbance at 808 nm is linearly dependent on the concentration (Fig. S3b), and the molar extinction coefficient of BP@lipid-PEG nanospheres was calculated to be 48.24 L mol-1 cm-1. The relative fluorescence quantum yield of BP@lipid-PEG nanospheres was determined to be 8% that of IR-26, which has been used as a standard reference fluorophore with an absolute quantum yield of 0.5% (Fig. 3d, e). We further investigated the fluorescence stability of BP@lipid-PEG nanospheres. The samples were diluted for measurement because of above mentioned concentration quenching effect (Fig. 2b, c). As shown in Fig. 2f, g, there is no big difference in their absorption with the extension of storage at room temperature, indicating surface modification can stabilize BP and prevent its from rapid degradation.47,48 But their fluorescence shows notable change, particularly for these at 1290 nm and 1607 nm. Under the ambient conditions (H2O, O2, and light), the photogenerated electrons in the conduction band of BP were trapped by O2 to form O2-, which in turn would react with P atom and lead to the quenching of fluorescence.45 Contrast to the notable change of fluorescence, their hydrodynamic sizes did not show big difference, whether these BP@lipid-PEG nanospheres were dispersed in pure H2O, 0.9% NaCl, PBS, or 10% FBS aqueous solution (Fig. S4). These results demonstrate that the fluorescence of BP@lipid-PEG nanospheres is very sensitive to the surrounding conditions. Under irradiation of NIR light, the photogenerated electrons in conduction bands of 10 / 29


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BP nanostructures can return to their valence bands through either radiative transition to generate fluorescence, or through nonradiative transition to produce heat. Previous reports have demonstrated that BP nanostructures can serve as an excellent photothermal transducer for PA imaging and photothermal therapy. We thereby studied the photothermal conversion property of BP@lipid-PEG nanospheres. As shown in Fig. S5a, the temperature of aqueous solutions of BP@lipid-PEG nanospheres was rapidly increased in contrast to pure water, and increased with the nanospheres concentration, which demonstrates their excellent photothermal conversion performance. In addition, the BP@lipid-PEG nanospheres exhibited great photothermal stability (Fig. 2h) during five cycles of heating and cooling treatments with an 808 nm laser. The fluorescence stability of BP@lipid-PEG nanospheres before and after 808 nm laser irradiation was investigated. Interestingly, their fluorescence from 900 to 1400 nm became much weaker, and the fluorescence from 1400 to 1650 nm did not show obvious change (Fig. 2i). To understand such changes caused by laser irradiation, the structure and optical properties of BP@lipid-PEG nanospheres were further analyzed. After five cycles of 808 nm laser irradiation, no obvious change in their hydrodynamic size and UV-visNIR absorption were observed (Fig. S5b, c). Their TEM image revealed that the shape of nanospheres became irregular, the surface lipid membrane was partially lost and the nanoparticles inside were looser from each other, in comparison with freshly prepared BP@lipid-PEG nanospheres before irradiation. These results demonstrate that their surface lipid-PEG membranes were changed to some extent during the photothermal 11 / 29



conversion due to their low glass transition temperature (41 ºC), which made BP-Chol nanoparticles expose to water and oxygen.17 The hydrogen bonds can be formed between water molecule and PxOy on the surface of BP nanoparticles, leading to the transfer of electrons between them and the quenching of fluorescence. Oxygen molecules can absorb electrons in the conduction bands of BP and transform into superoxide anions, which quenched the fluorescence. Moreover, the co-adsorption of oxygen and water molecules caused the indirect band gap of BP, which increased the energy loss and the quenching of fluorescence. 49-52 The influence of water and oxygen on the fluorescence of thin BP nanoparticles is stronger than their influence on thick nanoparticles. Therefore, the fluorescence in the range of 900 to 1400 nm became much weaker after laser irradiation, and the fluorescence from 1400 to 1650 nm remained the same as before. To further investigate the effect of surface modification on fluorescence of BP nanoparticles, we prepared dextran modified hydrophilic BP nanoparticles (referred to as BP-DEX) and measured their fluorescence.15 As shown in Fig. S5d, fluorescence of BP@lipid-PEG nanospheres is much stronger (5 times calculated by integral area ratio) than that of BP-DEX nanoparticles. The results demonstrate the significance of surface modification of BP nanoparticles with hydrophobic cholesterol. It should be noted that fluorescence of BP-Chol nanoparticles in CHCl3 solution is slightly stronger than that of its THF solution, but much weaker than that of BP@lipid-PEG nanospheres. 3.3 NIR-II fluorescence imaging It is known that NIR-II fluorescence possesses merit of deep penetration, different 12 / 29


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thicknesses of chicken breast slices (1 mm, 2 mm and 3 mm) were used as biological tissue to explore the penetration depth of fluorescence imaging acquired by using different wavelength filters. In Fig. 3a, a capillary (diameter = 0.41 mm) filled with BP@lipid-PEG nanospheres aqueous solution (400 μg mL-1) was closely covered with the chicken breast slice, and then irradiated with an 808 nm laser, the NIR-II fluorescence images were captured by using optical filters with different longpass wavelength of 810 nm, 1000 nm, 1250 nm, and 1400 nm, respectively. When the capillary was covered with 3 mm chicken slice, the fluorescence images captured with 1400 nm filter showed the sharpest contrast due to the lowest scattering and tissue autofluorescence beyond 1400 nm. Furthermore, we measured the cross-sectional fluorescence signal intensity along the yellow dotted line in the NIR-II fluorescence images captured with different filters by using Image J software (Fig. 3b). The FWHM (full width at half-maximum) of the peak fitted is broadened with the decrease of wavelength of optical filters, which can be attributed to the scattering of shortwavelength fluorescence and the biological tissue autofluorescence at short wavelength. The FWHM of the peak in fluorescence image captured with 1400 nm filter was 0.45 mm, which was very approximated to the diameter of capillary (0.41 mm). Therefore, the fluorescence of BP@lipid-PEG nanospheres beyond 1400 nm can be used for high contrast and high resolution NIR-II fluorescence bioimaging. To further assess performance of BP@lipid-PEG nanospheres for NIR-II fluorescence imaging, we intravenously injected an aqueous solution of BP@lipid-PEG nanospheres (150 μL, 1.3 mg mL-1) into normal nude mice anesthetized with 1.5% 13 / 29



isoflurane, and then captured NIR-II fluorescence images with the NIR-II Imaging System (808 nm laser, 1400 nm optical filter) before and post injection of 30 s, 5 min, 10 min, 20 min, 30 min, 1 h, 2 h, 4 h, 8 h, 16 h and 24 h, respectively (Fig. 4a). Before injection of BP@lipid-PEG nanospheres, it is hard to detect the NIR-II fluorescence signal in nude mouse. After injection, the surficial blood vessels and liver can be immediately observed due to the strong NIR-II fluorescence of nanospheres. To demonstrate the superiority of long wavelength to short wavelength in NIR-II imaging, we captured images at 30 s post injection by using 1400 nm and 1250 nm optical filters. As shown Fig. 4b, the enlarged part clearly shows the vessels in legs of the mouse, even for the small branched vessels labeled with yellow dotted Line 1. The FWHM values of two vessels are 0.37 mm and 0.49 mm, respectively. In contrast, these branched vessels (labeled with Line 1) are blurry in the image captured with 1250 nm optical filter, and their FWHM values are measured to be 0.90 mm and 0.80 mm (Fig. 4c). Due to the large size of BP@lipid-PEG nanospheres, they were quickly cleared from blood vessels and accumulated in the liver. The time-dependent variation of liver signal with nanospheres circulation was plotted in Fig. 4d, which shows that the NIR-II fluorescence signal in liver quickly reached the strongest at 5 min post injection, and then gradually decreased in 24 h. The signal intensity acquired at 24 h post injection was decreased to 47.6% of the maximum. Accompanied with the decrease of fluorescence signal in liver, the fluorescence signal of spleen was increased and achieved the highest at 24 h post injection. These results illustrate that BP@lipid-PEG 14 / 29


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nanospheres were quickly accumulated in liver after administration due to their large size, and then gradually metabolized into spleen. 3.4 Photoacoustic imaging The above results demonstrate that BP@lipid-PEG nanospheres exhibit great potential in in vivo NIR-II fluorescence imaging with high signal to noise ratio and low background. Since BP@lipid-PEG nanospheres could also convert NIR light into heat, their potential in photoacoustic (PA) imaging was assessed by a real-time multispectral photoacoustic tomographic imaging system. As shown in Fig. 5a, BP@lipid-PEG nanospheres aqueous solutions with different concentrations (0, 15.6, 31.3, 62.5, 125, 250 and 500 μg mL-1) were used for in vitro photoacoustic imaging. The signal intensity is linearly dependent on the concentration of BP@lipid-PEG nanospheres (Fig. S6), indicating that PA imaging can be used to semi-quantify bio-distribution of BP@lipidPEG nanospheres. Furthermore, normal nude mice anesthetized with 1.5% isoflurane were imaged before and post injection of 10 min, 20 min, 30 min, 1 h, 2 h, 4 h, 8 h, 24 h and 48 h (Fig. 5b). As shown in Fig. 5b, c, it is observed that the BP@lipid-PEG nanospheres were constantly accumulated in liver to achieve the maximum after 8 h post injection. After 24 h injection, the PA signal intensity in liver was decreased to 41.5% of the maximum, which is close to that in the NIR-II fluorescence imaging. After 48 h injection, the intensity in liver decreased to the level of pre-contrast image, which suggests that most BP@lipid-PEG nanospheres in the liver were metabolized. To further confirm the metabolism of BP@lipid-PEG nanospheres, the mouse was sacrificed to collect its heart, liver, spleen, lung, kidney, intestines and stomach for 15 / 29



observing with the NIR-II Imaging System (808 nm laser, 1250 nm optical filter). The weak signals of liver, lung and spleen support that most BP@lipid-PEG nanospheres were metabolized (Fig. 5e). The results suggest that PA imaging and NIR-II fluorescence real-time imaging can be used to study the bio-distribution of BP@lipidPEG nanospheres.

4. CONCLUSIONS In summary, we have successfully prepared BP@lipid-PEG nanospheres, and investigated their NIR-II fluorescence and application for bioimaging. The resultant BP@lipid-PEG nanospheres simultaneously exhibited broad NIR-II fluorescence from 900 nm to 1650 nm, and excellent photothermal conversion property and photothermal stability. Their NIR-II photoluminescence is very sensitive to water and oxygen, evidenced by drastic change of fluorescence after five cycles of laser irradiation. The surface hydrophobic cholesterol and lipid encapsulation play significant role in maintaining the NIR-II fluorescence of BP nanoparticles. The BP@lipid-PEG nanospheres were applied for in vivo NIR-II fluorescence imaging of nude mice, in which blood vessels, liver and spleen organs were clearly observed with a high signalto-noise ratio by using their emission beyond 1400 nm. The BP@lipid-PEG nanospheres were also used for in vivo photoacoustic imaging for semi-quantify their bio-distribution and pharmokinetics. Both NIR-II fluorescence imaging and PA imaging clearly show that BP@lipid-PEG nanospheres were gradually metabolized by

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liver in 48 h. Our work demonstrates black phosphorus has great potential for NIR-II fluorescence bioimaging due to their biodegradability and biocompatibility.

ASSOCIATED CONTENT Supporting Information This material is available free of charge via the Internet at http://pubs.acs.org.

ACKNOWLEDGEMENTS Z. Li acknowledges support from the National Key Research and Development Program of China (2018YFA0208800), National Natural Science Foundation of China (81471657, 81527901), the 1000 Plan for Young Talents, and Jiangsu Specially Appointed Professorship, and the Program of Jiangsu Innovative and Entrepreneurial Talents. The authors also are grateful for support from the Jiangsu Provincial Key Laboratory of Radiation Medicine and Protection, the Priority Academic Development Program of Jiangsu Higher Education Institutions (PAPD).

Received: ((will be filled in by the editorial staff)) Revised: ((will be filled in by the editorial staff)) Published online: ((will be filled in by the editorial staff))

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Figure 1. (a) Schematic diagram of BP@lipid-PEG nanospheres. (b) TEM image of BP-Chol nanoparticles. (c) AFM image of BP-Chol nanoparticles and statistical analysis of heights of BP-Chol nanoparticles in AFM image. (d) XRD patterns of BP and BP-Chol nanoparticles in comparison with standard pattern of BP. (e) TEM image and HRTEM image (inset) of BP@lipid-PEG nanospheres. (f) Diameter distribution of BP@lipid-PEG nanospheres shown in TEM image in Figure S2.

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Figure 2. (a) Optical images of an aqueous solution of BP@lipid-PEG nanospheres (1.3 mg mL-1) without and with irradiation by an 808 nm laser. (b) Emission spectra of BP@lipid-PEG nanospheres aqueous solutions with different concentrations under excitation by an 808 nm laser. (c) The dependence of fluorescence intensity at 1070 nm on particle concentration shown in (b). (d) UV–vis–NIR absorption spectra of BP@lipid-PEG nanospheres aqueous solution (68.2 µg mL-1) and IR-26 dichloroethane solution. (e) Emission spectra of BP@lipid-PEG nanospheres and IR-26 shown in (d). (f) UV–vis–NIR absorption spectra and digital photographs of BP@lipid-PEG nanospheres after exposure to air for different days. (g) Emission spectra excited with an 808 nm laser of BP@lipid-PEG nanospheres solution shown in (f). (h) Heating profiles of an aqueous solution of BP@lipid-PEG nanospheres under irradiation by an 24 / 29


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808 nm laser with a power density of 1 W cm-2 for five cycles. (i) Emission spectra of BP@lipid-PEG nanospheres solution before and after five cycles of 808 nm laser irradiation. (j) TEM image of BP@lipid-PEG nanospheres after five cycles of 808 nm laser irradiation.

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Figure 3. (a) Photoluminescence images of a capillary (diameter = 0.41 mm) filled with BP@lipid-PEG nanospheres aqueous solution (400 μg mL-1) and covered with chicken breast slice with different thickness, captured by using different wavelength optical filters (i.e. longpass 1400 nm, 1250 nm, 1000 nm, and 810 nm). (b) Cross-sectional fluorescence signal intensity profiles along the yellow dotted line shown in (a). Scale bar = 5 mm.

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Figure 4. (a) In vivo NIR-II fluorescence images of mice collected at different time intervals by using 1400 nm optical filter, after intravenous injection of BP@lipid-PEG nanospheres aqueous solutions. (b) Enlarged part of the image acquired at 30 s post injection with different optical filters (1400 nm, 1250 nm). (c) Cross-sectional fluorescence signal intensity profiles along the yellow dotted Line 1 and Line 2. (d) Time-dependent variation of signal intensity of the liver images captured with 1400 nm longpass filter. Scale bar = 5 mm.

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Figure 5. (a) In-vitro photoacoustic images of BP@lipid-PEG nanospheres aqueous solutions. (b) In vivo photoacoustic images of liver at different time intervals after intravenous injection of BP@lipid-PEG nanospheres aqueous solutions. (c) Variation of PA signals of liver with time after injection of BP@lipid-PEG nanospheres aqueous solutions. (d) Photograph of heart, liver, spleen, lung, kidney, intestines and stomach of mice post injection of 48 h. (e) NIR-II fluorescence imaging of organs shown in (d).

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Cholesterol-Modified Black Phosphorus Nanospheres for the First NIR

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